BACKGROUND
[0001] The synthesis of oligonucleotide probes on
in situ synthesized arrays, such as by photolithography, can result in a population of incomplete
or truncated probe sequences which accompany the probe sequences synthesized at the
full desired or intended length ("full-length" probes). The presence of such truncated
probe sequences can have a detrimental effect on array performance, especially in
arrays requiring enzymatic addressing of the free probe terminus, such as polymerase
extension reactions or ligation reactions.
[0002] In contrast, oligonucleotide probes immobilized on bead arrays (e.g., Illumina) and
other spotted arrays are commonly attached to their substrates via an amine or other
functional group synthetically attached to the 5' end of the probe. In this way, only
full-length sequences are immobilized, and truncations or other defects associated
with an exposed free 3' end are reduced or virtually eliminated.
SUMMARY
[0003] It can be desirable to selectively remove truncated probe sequences post-synthesis
from among the probes on
in situ synthesized arrays, such as those fabricated with photolithography. The present disclosure
provides processes for accomplishing this selective removal of truncated sequences,
while simultaneously inverting the orientation of the probe sequence such that probe
sequences synthesized from the 3'-end are converted to probe sequences attached to
the substrate via their 5'-end.
[0004] In particular, this disclosure includes probe inversion processes for in situ synthesized
arrays which can use universal linkers and commercially available building blocks
and reagents. These can include incorporation of a universal cleavable linker phosphoramidite
for use in releasing free 3'-OH termini, incorporation of branched linkers with orthogonally
protected, addressable functional groups for oligonucleotide synthesis and post-synthesis
circularization, more efficient crosslinking chemistries for circularization steps
utilizing commercially available reagents, and other improvements. Previous processes
attempting probe inversion on in situ synthesized arrays involved a large number of
special linkers, building blocks and reagents, which can make it impractical to use
for large scale manufacturing of in situ synthesized arrays.
[0005] An aspect of the present disclosure provides a method, comprising: providing a substrate
with a plurality of branched linkers coupled to said substrate, wherein said substrate
comprises a plurality of hydroxyalkyl groups, wherein each branched linker comprises
(i) a first branch comprising a first alkyne and (ii) a second branch, wherein said
second branch comprises a first cleavable linker coupled to a 3' end of a first oligonucleotide,
and wherein a 5' end of said first oligonucleotide is coupled to a first azide group;
and circularizing said first oligonucleotide by reacting said first azide group with
a second alkyne, wherein said second alkyne is said first alkyne or a neighboring
alkyne; wherein efficiency of said circularization increases when said second branch
further comprises a capping moiety.
[0006] In some embodiments of aspects provided herein, said plurality of branched linkers
are coupled to said substrate via said plurality of hydroxyalkyl groups. In some embodiments
of aspects provided herein, said plurality of branched linkers coupled to said substrate
via a first intermediate selected from the group consisting of the structures shown
in
FIG. 2A, FIG. 2B, and
FIG. 2C. In some embodiments of aspects provided herein, said second alkyne is said first
alkyne. In some embodiments of aspects provided herein, said first cleavable linker
becomes part of said branched linker via a second intermediate selected from the group
consisting of the structures shown in
FIG. 3A, FIG. 3B, and
FIG. 3C. In some embodiments of aspects provided herein, the ratio between said cleavable
linker and said first alkyne is about 5, about 4, about 3, about 2, about 1, about
0.5, about 0.33, about 0.25, about 0.2, or about 0.1. The method of claim 1, the method
further comprises (c) cleaving said first cleavable linker, thereby decoupling said
3' end of said first oligonucleotide from said second branch. In some embodiments
of aspects provided herein, said cleaving comprises de-protection with a base. In
some embodiments of aspects provided herein, said said efficiency is at least 70%,
at least 80%, at least 90%, or at least 95%. In some embodiments of aspects provided
herein, another second branch comprises a second cleavable linker coupled to a 3'
end of a second oligonucleotide, wherein said second oligonucleotide is shorter than
said first oligonucleotide, and wherein said cleaving releases said second oligonucleotide
from said substrate. In some embodiments of aspects provided herein, said cleaving
releases at least 90% of said second oligonucleotide from said substrate.
[0007] Another aspect of the present disclosure provides a method, comprising: providing
a substrate;
attaching a plurality of branched linkers to said substrate, wherein said branched
linkers comprise (i) a first branch comprising an alkyne and (ii) a second branch;
attaching a cleavable linker to a first group of said second branches; attaching a
capping moiety to a second group of said second branches; synthesizing a first oligonucleotide
on said cleavable linker in 3' to 5' orientation, said first oligonucleotide comprising
(i) a 3' end coupled to said second branch via said cleavable linker and (ii) a 5'
end coupled to an azide group; and circularizing said first oligonucleotide by reacting
said azide group with said alkyne, thereby coupling said 5' end of said first oligonucleotide
to one of said first branches.
[0008] In some embodiments of aspects provided herein, the ratio of said first group of
second branches to said second group of second branches is between about 5:1 to about
1:5. In some embodiments of aspects provided herein, the ratio of said first group
of second branches to said second group of second branches is about 5:1, about 4:1,
about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, or about 1:5. In
some embodiments of aspects provided herein, the ratio of said first group of second
branches to said second group of second branches is about 1 or less. In some embodiments
of aspects provided herein, wherein said capping moiety is attached via an intermediate
of capping phosphoramidite. In some embodiments of aspects provided herein, said substrate
comprises a plurality of hydroxyalkyl groups. In some embodiments of aspects provided
herein, said branched linker is coupled to said substrate via said plurality of hydroxyalkyl
groups. In some embodiments of aspects provided herein, the method further comprises
(c) cleaving said cleavable linker, thereby de-coupling said 3' end of said first
oligonucleotide from said first group of said second branch. In some embodiments of
aspects provided herein, a second oligonucleotide is attached to a third group of
said second branch, wherein said second oligonucleotide lacks a covalent bond to said
first branch, wherein said cleaving releases said second oligonucleotide from said
substrate. In some embodiments of aspects provided herein, the substrate comprises
controlled core glass. In some embodiments of aspects provided herein, said synthesizing
comprises photolithographic synthesis.
[0009] Another aspect of the present disclosure provides a system, comprising: a substrate
comprising a plurality of hydroxyalkyl groups; and a plurality of branched linkers
coupled to said plurality of hydroxyalkyl groups, wherein each said branched linker
comprises (i) a first branch comprising a first alkyne and (ii) a second branch, wherein
said second branch comprises a first cleavable linker and a capping moiety, wherein
said first cleavable linker is coupled to a 3' end of a first oligonucleotide, and
wherein a 5' end of said first oligonucleotide is coupled to a first azide group.
[0010] In some embodiments of aspects provided herein, said first azide group reacts with
a second alkyne to circularize said first oligonucleotide, wherein said second alkyne
is said first alkyne or a neighboring alkyne. In some embodiments of aspects provided
herein, the ratio between said cleavable linker and said capping moiety is about 10,
about 5, about 4, about 3, about 2, about 1, about 0.5, about 0.33, about 0.25, about
0.2, or about 0.1. In some embodiments of aspects provided herein, said plurality
of branched linkers coupled to said substrate via a first intermediate selected from
the group consisting of the structures shown in
FIG. 2A, FIG. 2B, and
FIG. 2C. In some embodiments of aspects provided herein, said first cleavable linker becomes
part of said branched linker via a second intermediate selected from the group consisting
of the structures shown in
FIG. 3A, FIG. 3B, and
FIG. 3C. In some embodiments of aspects provided herein, said capping moiety is acyl, dialkoxyphosphoryl,
alkyl, alkoxycarbonyl, or dialkyaminocarbonyl. In some embodiments of aspects provided
herein, said first cleavable linker is cleavable by with a base, for example, ammonia,
methyl amine, 1,2-diaminoethane, and potassium carbonate.
[0011] Another aspect of the present disclosure provides a method, comprising: providing
a substrate with a plurality of branched linkers coupled to said substrate, wherein
each branched linker comprises (i) a first branch comprising a first alkyne and (ii)
a second branch, wherein said second branch comprises a cleavable linker coupled to
a 3' end of a first oligonucleotide, and wherein a 5' end of said first oligonucleotide
is coupled to an azide group; reacting said azide group with a second alkyne to circularize
said first oligonucleotide, said reaction being catalyzed by a Cu(I) catalyst in a
buffer at room temperature, said second alkyne being said first alkyne or a neighboring
alkyne; and (c) cleaving said cleavable linker, thereby inverting said first oligonucleotide.
[0012] In some embodiments of aspects provided herein, said second alkyne is said first
alkyne. In some embodiments of aspects provided herein, the method further comprises:
(d) providing a second oligonucleotide complementary to at least part of inverted
first oligonucleotide obtained in (c), wherein said second oligonucleotide leaves
a 3' overhang after hybridization with said inverted first oligonucleotide; and (e)
extending said second oligonucleotide with a polymerase and fluorescently labeled
dNTP, thereby confirming said inversion of said first oligonucleotide.
[0013] Additional aspects and advantages of the present disclosure will become readily apparent
to those skilled in this art from the following detailed description, wherein only
illustrative embodiments of the present disclosure are shown and described. As will
be realized, the present disclosure is capable of other and different embodiments,
and its several details are capable of modifications in various obvious respects,
all without departing from the disclosure. Accordingly, the drawings and description
are to be regarded as illustrative in nature, and not as restrictive.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The novel features of the invention are set forth with particularity in the appended
claims. A better understanding of the features and advantages of the present invention
will be obtained by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention are utilized, and
the accompanying drawings of which:
FIG. 1A shows an exemplary process for inverting a probe.
FIG. 1B shows exemplary intermediates for branched linker (left) and cleavable linker (center),
and bromohexyl phosphoramidite (right).
FIG. 1C shows an exemplary oligonucleotide (SEQ ID NO: 1) used for probe inversion incorporating
intermediates in FIG. 1B.
FIG. 2A shows an exemplary intermediate leading to branched linker.
FIG. 2B shows an exemplary intermediate leading to branched linker.
FIG. 2C shows an exemplary intermediate leading to branched linker.
FIG. 3A shows an exemplary intermediates leading to cleavable linker (CL).
FIG. 3B shows an exemplary intermediates leading to cleavable linker (CL).
FIG. 3C shows an exemplary intermediates leading to cleavable linker (CL).
FIG. 4A shows an exemplary HPLC profile of non-inverted oligonucleotides.
FIG. 4B shows an exemplary HPLC profile of inverted oligonucleotides without capping phosphoramidites.
FIG. 4C shows an exemplary HPLC profile of inverted oligonucleotides with a 1:1 ratio of
cleavable linkers to capping phosporamidites.
FIG. 4D shows an exemplary HPLC profile of inverted oligonucleotides with a 1:2 ratio of
cleavable linkers to capping phosporamidites.
FIG. 5A shows an exemplary mass spectrograph of non-inverted oligonucleotides.
FIG. 5B shows an exemplary mass spectrograph of inverted oligonucleotides.
FIG. 6A shows an exemplary fluorescence image of a control chip with no oligonucleotides.
FIG. 6B shows an exemplary fluorescence image of a control chip with non-inverted oligonucleotides.
FIG. 6C shows an exemplary fluorescence image of a chip with inverted oligonucleotides.
FIG. 7A shows an exemplary fluorescence image of a chip with inverted oligonucleotides prior
to an extension reaction.
FIG. 7B shows an exemplary fluorescence image of a chip with inverted extended oligonucleotides.
FIG. 7C shows an exemplary fluorescence image of a chip with non-inverted oligonucleotides
prior to an extension reaction.
FIG. 7D shows an exemplary fluorescence image of a chip with non-inverted oligonucleotides
after an extension reaction.
FIG. 8A shows a schematic of an array of oligonucleotides with different sequence permutations
of -NNT-3' (SEQ ID NOS 4 and 5, respectively, in order of appearance).
FIG. 8B shows an exemplary fluorescence image of a chip with inverted oligonucleotides after
an extension reaction.
FIG. 9A shows a schematic of an array of oligonucleotides with different sequence permutations
of -NN-3' (SEQ ID NOS 6 and 5, respectively, in order of appearance).
FIG. 9B shows an exemplary fluorescence image of a chip with inverted oligonucleotides after
an extension reaction.
FIG. 10 shows an image of alignment marks on a wafer with inverted nucleotides hybridized
with Cy3-labeled complementary DNA.
FIG 11A shows an image of incorporation of dGTP nucleotides in a single-base extension.
FIG 11B shows an image of incorporation of dTTP nucleotides in a single-base extension.
FIG 11C shows an image of incorporation of dATP nucleotides in a single-base extension.
FIG 11D shows an image of incorporation of dCTP nucleotides in a single-base extension.
FIG 12A shows an image of incorporation of dGTP nucleotides at 3' postion of inverted wafer
chip in a single-base extension.
FIG 12B shows an image of incorporation of dTTP nucleotides at 3' position of inverted wafer
chip in a single-base extension (inset: enlarged portion of actual image showing array
features).
FIG 12C shows an image of incorporation of dATP nucleotides at 3' position of inverted wafer
chip in a single-base extension.
FIG 12D shows an image of incorporation of dCTP nucleotides at 3' position of inverted wafer
chip in a single-base extension.
DETAILED DESCRIPTION
[0015] The present disclosure provides processes for the circularization and inversion of
in situ synthesized oligonucleotide probes. Particular circularization chemistries
are described, including click chemistry reactions using bromo or iodide groups. Such
reactions can be used to bind the 5' ends of oligonucleotides to surface-bound linker
molecules; circularized oligonucleotides can then have their 3' ends released, thereby
inverting the oligonucleotides. The processes disclosed herein can also reduce or
eliminate truncated oligonucleotide probes, which do not contain the full synthesized
oligonucleotide sequence, while preserving full-length oligonucleotide probes, which
do contain the full synthesized oligonucleotide sequence. For example, full-length
oligonucleotides can be circularized prior to release of the 3' ends, while non-full-length
oligonucleotides remain un-circularized and therefore are removed from the surface
upon release of the 3' ends.
[0016] The term "oligonucleotide" can refer to a nucleotide chain. In some cases, an oligonucleotide
is less than 200 residues long, e.g., between 15 and 100 nucleotides long. The oligonucleotide
can comprise at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,
40, 45, or 50 bases. The oligonucleotides can be from about 3 to about 5 bases, from
about 1 to about 50 bases, from about 8 to about 12 bases, from about 15 to about
25 bases, from about 25 to about 35 bases, from about 35 to about 45 bases, or from
about 45 to about 55 bases. The oligonucleotide (also referred to as "oligo") can
be any type of oligonucleotide (e.g., a primer). Oligonucleotides can comprise natural
nucleotides, non-natural nucleotides, or combinations thereof.
[0017] The term "click chemistry" can refer to reactions that are modular, wide in scope,
give high yields, generate only inoffensive byproducts, such as those that can be
removed by nonchromatographic methods, and are stereospecific (but not necessarily
enantioselective). An exemplary click chemistry reaction is the Huisgen 1,3-dipolar
cycloaddition of an azide and an alkynes, i.e., Copper-catalyzed reaction of an azide
with an alkyne to form a 5-membered heteroatom ring, known as a Cu(I)-Catalyzed Azide-Alkyne
Cycloaddition (CuAAC).
[0018] The term "click chemistry moiety" can be a terminal alkyne.
[0019] The term "about" as used herein refers to +/- 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,
or 1% of the designated amount.
[0020] The term "circularize" or "circularization" as used herein refers to the structure
of an oligonucleotide with both of its ends attached to the substrate or support via
covalent bonds.
[0021] Genetic information can be utilized in a myriad of ways with the advent of rapid
genome sequencing and large genome databases. One of such applications is oligonucleotide
arrays. The general structure of an oligonucleotide array, or commonly referred to
as a DNA microarray or DNA array or a DNA chip, is a well-defined array of spots or
addressable locations on a surface. Each spot can contain a layer of relatively short
strands of DNA called "probe" or "capture probe" (e.g.,
Schena, ed., "DNA Microarrays A Practical Approach," Oxford University Press;
Marshall et al. (1998) Nat. Biotechnol. 16:27-31). There are at least two technologies for generating arrays. One is based on photolithography
(e.g. Affymetrix) while the other is based on robot-controlled ink jet (spotbot) technology
(e.g., Arrayit.com). Other methods for generating microarrays are known and any such
known method may be used herein.
[0022] Generally, an oligonucleotide (probe or capture probe) placed within a given spot
in the array is selected to bind at least a portion of a nucleic acid or complimentary
nucleic acid of a target nucleic acid. An aqueous sample is placed in contact with
the array under the appropriate hybridization conditions. The array is then washed
thoroughly to remove all non-specific adsorbed species. In order to determine whether
or not the target sequence was captured, the array can be "developed" by adding, for
example, a fluorescently labeled oligonucleotide sequence that is complimentary to
an unoccupied portion of the target sequence. The microarray is then "read" using
a microarray reader or scanner, which outputs an image of the array. Spots that exhibit
strong fluorescence are positive for that particular target sequence.
[0023] A probe can comprise biological materials deposited so as to create spotted arrays.
A probe can comprise materials synthesized, deposited, or positioned to form arrays
according to other technologies. Thus, microarrays formed in accordance with any of
these technologies may be referred to generally and collectively hereafter for convenience
as "probe arrays." The term "probe" is not limited to probes immobilized in array
format. Rather, the functions and methods described herein can also be employed with
respect to other parallel assay devices. For example, these functions and methods
may be applied when probes are immobilized on or in beads, optical fibers, or other
substrates or media.
[0024] In methods and systems of the present disclosure, probes can be attached to a solid
substrate. Probes can be bound to a substrate directly or via a linker. Linkers can
comprise, for example, amino acids, polypeptides, nucleotides, oligonucleotides, or
other organic molecules that do not interfere with the functions of probes.
[0025] The solid substrate can be biological, non-biological, organic, inorganic, or a combination
of any of these. The substrate can exist as one or more particles, strands, precipitates,
gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates,
slides, or semiconductor integrated chips, for example. The solid substrate is can
be flat or can take on alternative surface configurations. For example, the solid
substrate can contain raised or depressed regions on which synthesis or deposition
takes place. In some examples, the solid substrate can be chosen to provide appropriate
light-absorbing characteristics. For example, the substrate can be a polymerized Langmuir
Blodgett film, functionalized glass (e.g., controlled pore glass), silica, titanium
oxide, aluminum oxide, indium tin oxide (ITO), Si, Ge, GaAs, GaP, SiO
2, SiN
4, modified silicon, the top dielectric layer of a semiconductor integrated circuit
(IC) chip, or any one of a variety of gels or polymers such as (poly)tetrafluoroethylene,
(poly)vinylidenedifluoride, polystyrene, polycarbonate, polydimethylsiloxane (PDMS),
polymethylmethacrylate (PMMA), polycyclicolefins, or combinations thereof.
[0026] Solid substrates can comprise polymer coatings or gels, such as a polyacrylamide
gel or a PDMS gel. Gels and coatings can additionally comprise components to modify
their physicochemical properties, for example, hydrophobicity. For example, a polyacrylamide
gel or coating can comprise modified acrylamide monomers in its polymer structure
such as ethoxylated acrylamide monomers, phosphorylcholine acrylamide monomers, betaine
acrylamide monomers, and combinations thereof.
[0027] DNA microarrays can be fabricated using spatially-directed
in situ synthesis or immobilization of pre-synthesized oligonucleotides. In both cases, synthesis
of the oligonucleotides typically proceeds with the addition of monomers in the 3'-to-5'
direction, using standard 3'-phosphoramidite reagents and solid-phase synthesis protocols
(e.g.,
M. Egli, et al., ed. "Current Protocols in Nucleic Acid Chemistry," John Wiley & Sons). The main impurities are truncated, partial-length sequences resulting from incomplete
monomer coupling and, to a lesser extent, depurination reactions.
[0028] On the one hand, fabricating arrays of pre-synthesized oligonucleotide probes typically
involves covalent attachment of the oligonucleotides to a substrate through the 5'-terminus,
via a reactive modifier which is added to the end when the oligonucleotides are synthesized
on high-throughput synthesizers (see
S. J. Beaucage, et al., Curr. Med. Chem. 2001, 8, 1213-44). This ensures that the probes which are attached to the support are primarily full-length
sequences, since truncated sequences are capped and rendered non-reactive during synthesis
(
Brown T and Brown T, Jr. (2005-2015) Solid-phase oligonucleotide synthesis. [Online]
Southampton, UK, ATDBio. <http://www.atdbio.com/content/17/Solid-phase-oligonucleotide-synthesis> [Accessed August 9, 2016]).
[0029] A significant advantage of the present disclosure is that the 3'-hydroxyl group of
the oligonucleotide probe is "distal" to the substrate, and freely available for enzymatic
reactions, such as template-directed polymerase-catalyzed chain extension and ligation;
and this character can be exploited to carry out very sensitive and specific assays
for detecting and quantitating genetic polymorphisms (
K. Lindroos, et al., Nucleic Acids Res. 2001, 29, e69;
Gunderson KL, et al., Nature Genetics 2005, 37, 549-54).
[0030] On the other hand, DNA microarrays can also be fabricated using
in situ synthesis of sequences directly on the support. In this case, sequences are "printed"
in a highly parallel fashion by spatially-directing the synthesis using inkjet (
T. R. Hughes, et al., Nature Biotechnol 2001,19,342-7;
C. Lausted, et al., Genome Biol 2004, 5, R58), photolithographic technologies (
A.C. Pease, et al., Proc Natl Acad Sci USA 1994, 91, 5022-6;
G. McGall, et al., Proc Natl Acad Sci USA 1996; 93:13555-60;
S. Singh-Gasson, et al., Nature Biotechnol 1999, 17, 974-8;), or electrochemical techniques (
PLoS ONE 2006, 1, e34;
B. Y. Chow, et al., Proc Natl Acad Sci USA 2009, 106, 15219-24). Here too, synthesis proceeds 3' to 5' direction (solid-phase oligonucleotide synthesis
in the 5'-to-3' direction, while feasible, is much less efficient and economical,
providing lower yields and product purity). However, the resulting probes are attached
to the substrate at the 3'-terminus, and any truncated sequence impurities which arise
during the synthesis remain on the support, which may be a particular issue in the
case of photolithographic synthesis (
J. Forman, et al., Molecular Modeling of Nucleic Acids, Chapter 13, p. 221, American
Chemical Society (1998) and
G. McGall, et al., J. Am. Chem. Soc. 119:5081-5090 (1997)). As a result, polymerase-based extension assays normally are not feasible using
arrays which are made this way.
[0031] Despite the above limitation, photolithographic synthesis is a highly attractive
means of fabricating very high-density DNA arrays, as it is capable of exceeding 10
million arrayed sequences per cm
2 (
A. R. Pawloski, et al., J Vac Sci Technol B 2007, 25, 2537-46), and is highly scalable in a manufacturing setting.
[0032] Thus, it is highly desirable to develop an effective method of inverting the sequences
on such probe arrays.
[0033] The plurality of probes can be located in one or more addressable regions (spots,
locations, etc.) on a solid substrate, herein referred to as "pixels." In some cases,
a solid substrate comprises at least about 2, 3, 4, 5, 6, or 7-10, 10-50, 50-100,
100-500, 500-1,000, 1,000-5,000, 5,000-10,000, 10,000-50,000, 50,000-100,000, 100,000-500,000,
500,000-1,000,000 or over 1,000,000 pixels with probes. In some cases, a solid substrate
comprises at most about 2, 3, 4, 5, 6, or 7-10, 10-50, 50-100, 100-500, 500-1,000,
1,000-5,000, 5,000-10,000, 10,000-50,000, 50,000-100,000, 100,000-500,000, 500,000-1,000,000
or over 1,000,000 pixels with probes. In some cases, a solid substrate comprises about
2, 3, 4, 5, 6, or 7-10, 10-50, 50-100, 100-500, 500-1,000, 1,000-5,000, 5,000-10,000,
10,000-50,000, 50,000-100,000, 100,000-500,000, 500,000-1,000,000 or over 1,000,000
pixels with probes.
[0034] In some cases it is useful to have pixels which do not contain probes. Such pixels
can act as control spots in order to increase the quality of the measurement, for
example, by using binding to the spot to estimate and correct for non-specific binding.
In some cases, the density of the probes can be controlled to either facilitate the
attachment of the probes or enhance the ensuing detection by the probes.
[0035] In some examples, it is useful to have redundant pixels which have identical probe
sequences to another pixel but physically may not be adjacent or in proximity to the
other pixel. The data acquired by such probe arrays may be less susceptible to fabrication
non-idealities and measurement errors.
[0036] In some cases, labels are attached to the probes within the pixels, in addition to
the labels that are incorporated into the targets. In such systems, captured targets
can result in two labels coming into intimate proximity with each other in the pixel.
As discussed before, interactions between specific labels can create unique detectable
signals. For example, when the labels on the target and probe, respectively, are fluorescent
donor and acceptor moieties that can participate in a fluorescent resonance energy
transfer (FRET) phenomenon, FRET signal enhancement or signal quenching can be detected.
Synthesis of Inverted Oligonucleotides
[0037] FIG. 1A shows an exemplary process for inversion of probes in an
in situ synthesized probe array and removal of truncated probe sequences. First, a synthesis
substrate is prepared, comprising available surface groups such as hydroxyl groups,
as shown in
FIG. 1A. These hydroxyl groups are part of hydroxyalkyl groups on the synthesis substrate
or support. For example, a surface may be modified with reagents, e.g., hydroxyalkyltrialkoxysilane,
to provide hydroxyalkyl groups available for the branched liker to attach. Alternatively,
a polymer thin film can be applied to the surface of the synthesis substrate or support,
wherein the polymer contains free hydroxyl groups for attachment. Furthermore, a support
that is compatible with DNA synthesis reagent and processing conditions and that contains
hydroxyalkyl groups can be used for the attachment of the branched linker.
[0038] A branched linker (e.g., ALK, in
FIG. 1B) can then be added to the surface of the synthesis substrate or support via all kinds
of intermediates to introduce an alkyne on one position/branch and a DMT-protected
hydroxyl group (to attach an oligonucleotide later) on another position/branch. The
above-mentioned intermediates for the introduction of branched linkers include but
are not limited to, a branched linker amidite intermediate (e.g., alkyne-modifier
serinol phosphoramidite in
FIG. 1B (left panel) and
FIG. 2C), or intermediates as shown in
FIG. 2A and
FIG. 2B.
[0039] Turning now to
FIGS. 2A and
2B, levulinyl (LEV) is a protecting group for hydroxyl groups and can be specifically
removed using a reagent containing hydrazine hydrate, acetic acid and pyridine (e.g.,
with 0.5 M hydrazine hydrate in 1:1 pyridine/acetic acid). 4,4'-Dimethoxytrityl (DMT)
is another hydroxyl protecting group that can be removed under acidic conditions.
2-Cyanoethyl (CE) is a phosphite/phosphate protection and can be removed under basic
conditions. The molecules shown in
FIGS. 2A, 2B and
2C are commercially available from Glen Research (Sterling, VA 20164).
[0040] Intermediates for the introduction of the branched linkers, for example, those shown
in
FIG. 2A and
FIG. 2B, can not only react with surface hydroxyalkyl groups to attach to the support, but
also have two orthogonally protected hydroxyl groups to allow the introduction of
azide-attached oligonucleotide, alknyne, or a capping moiety. For example, a few additional
steps are needed to attach an alkyne group to the first branch of the branched linker.
First, phosphoramidite intermediates in
FIG. 2A and
FIG. 2B are coupled to the surface hydroxyalkyl groups on the support, followed by an oxidation
of the resulting phosphite to phosphate. Then the LEV protection group is selectively
removed without affecting the DMT protection group to reveal a hydroxyl group. An
alkyne-containing phosphoramidite, for example, Compound A, is then coupled to the
unmasked hydroxyl group to complete the synthesis of the first branch with an alkyne.
Oxidation of the phosphite to phosphate can be carried out after the reaction of Compound
A with the surface hydroxyalkyl groups.
[0041] Alternatively, alkyne-modifier serinol phosphoramidite in
FIG. 2C can be coupled to the support directly. Because the alkyne group is preinstalled
in this intermediate, no extra step is needed to introduce an alkyne group to the
first branch.
[0042] After installation of the first branch containing an alkyne group, the DMT protection
group can be removed to provide another hydroxyl group as a handle for the attachment
of a cleavable linker, followed by the attachment of an oligonucleotide. Oligonucleotides
can be introduced by any oligonucleotide synthesis method in a stepwise manner. For
each round of oligonucleotide synthesis, a capping step is performed to cap a free
hydroxyl group after the extension (no extension of the oligonucleotide chain on this
free hydroxyl group) with an acetyl group or other suitable capping molecule after
the phosphorylation. In this way capped failure sequences are prevented from participating
in the rest of the chain elongation for the targeted oligonucleotide. Thus, these
caped failure sequences are truncated oligonucleotides as shown in
FIG. 1A. At the final step of the oligonucleotide chain extension, the unmasked 5' hydroxyl
group of the last nucleic acid can be capped with an azide containing tail. This step
can be achieved by first introducing a linker with a leaving group at the end. The
leaving group can be Cl, Br, I, mesylate, tosylate, or triflate. Then replace the
leaving group with an azide. An example of bromide to azide transformation is shown
on the second row of
FIG. 1A.
[0043] As described above, the linkers can comprise a post-synthesis reactive group, such
as for click chemistry. For example, an alkyne group can be used, which can be stable
during DNA synthesis of the oligonucleotides, and therefore would not require the
use of protection/de-protection strategies. Branched linkers and cleavable inkers
having phosphoramidite moieties, can be added to the synthesis substrate or support
to provide access points for the later introduction of oligonucleotides using standard
DNA synthesis protocols. In some cases, linkers can be added to the synthesis substrate
using standard DNA synthesis protocols with extended coupling times (e.g., 3 minutes).
[0044] Specifically, after a free hydroxyl group is formed, a cleavable linker amidite intermediate
(CL) can be incorporated into the branched linker, such as the cleavable linkers shown
in
FIG. 1B (middle panel),
FIG. 3A, FIG. 3B, and
FIG. 3C. Cleavable linkers can be added to the synthesis substrate using standard DNA synthesis
protocols. In some cases, cleavable linkers can be added to the synthesis substrate
using standard DNA synthesis protocols with extended coupling times (e.g., 3 minutes).
[0045] Standard oligonucleotide probe synthesis can then be conducted on the cleavable linker
via its free hydroxyl group to synthesize probe sequences. The synthesized probe sequences
can comprise full-length probe sequences and truncated probe sequences. Full-length
probe sequences can comprise a linker group on the 5' end with a bromo (e.g., bromine)
group (see, e.g.,
FIG. 1A) or an iodide group or other leaving groups at one end. For example, the last coupling
of a DNA synthesis protocol can be performed with 5'-bromohexyl phosphoramidite to
provide a bromine group at the 5' end of the synthesized oligonucleotides. In some
cases, full-length probe sequences can comprise an -OH group on the 5' end, and the
-OH group can react with reagents to add a linker with desired leaving group (such
as Br, I, tosylate, mesylate, and triflate) on its end. These leaving groups, on the
5' ends of full-length probe sequences, can then be converted to other groups, such
as azide groups. For example, a solution of sodium azide and sodium iodide in DMF
can be used to react synthesized oligonucleotides with a leaving group on its 5' end
to provide azide groups. In some cases, the synthesis substrate can be washed (e.g.,
with acetonitrile) before and/or after reaction with azide groups.
[0046] In one example, controlled pore glass (CPG) beads are used as the synthesis substrate,
and oligonucleotide probes are synthesized on cleavable linkers attached to the substrate.
The beads are washed in a column with acetonitrile and dried under nitrogen gas. The
washed beads are then added to a solution of 13 milligrams (mg) sodium azide and 30
mg sodium iodide in 2 milliliters (mL) of dry DMF and allowed to react for 1 hour
at 65 °C. The reaction mixture is then cooled and solvent is removed with a pipette,
and the beads are washed twice with 2 mL of DMF and 2 mL of acetonitrile. Then, the
beads are transferred to a column and dried under nitrogen gas. The 5' terminal hydroxyl
group of oligonucleotides can also be converted into azide-containing linker group
following a reported protocol published by
Eric T Kool (J Org Chem. 2004, 69, 2404-2410).
[0047] Azide groups on the ends of full-length probe sequences can be circularized to another
branch of the same or different branched linker, for example, as shown in the third
row in
FIG. 1A. Circularization can be accomplished through click chemistry (e.g., Cu(I) click chemistry
or Cu
+ click chemistry). Catalyst for the click chemistry can be Cu(I) salts, or Cu(I) salts
made
in situ by reducing Cu(II) reagent to Cu(I) reagent with a reducing reagent (
Pharm Res. 2008 Oct; 25(10): 2216-2230). Known Cu(II) reagents for the click chemistry are commercially available, including
but not limited to Cu(II)-(TBTA) complex and Cu(II) (THPTA) complex. TBTA, which is
tris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, also known as tris-(benzyltriazolylmethyl)amine,
is a stabilizing ligand for Cu(I) salts. THPTA, which is tris-(hydroxypropyltriazolylmethyl)amine,
is another stabilizing agent for Cu(I). Circularization can also be accomplished using
copper-free click DNA ligation, such as by the ring-strain promoted alkyne-azide cycloaddition
reaction (see, e.g.,
Chem. Commun., 2011, 47:6257-6259 or
Nature, 2015, 519(7544):486-90).
[0048] Circularization can be conducted such that only full-length probe sequences are circularized.
The cleavable linker can then be cleaved, for example by standard de-protection with
a base, including but not limited to, ammonia, methyl amine, 1,2-diaminoethane, and
potassium carbonate. Cleavage of the cleavable linker can release the 3'-OH terminus
of the probe sequences, releasing truncated probe sequences and inverting circularized
full-length probe sequences as shown in the third row of
FIG. 1A.
[0049] Due to the sizes of and steric constraints placed on the full-length probe sequences,
the efficiency and the location of the click chemistry reaction between the alkyne
and the azide can vary. In one embodiment, the circularization forms covalent bonds
between an alkyne group and an azide group, both of which are attached to the same
branched linker. In another embodiment, the circularization forms covalent bonds between
an alkyne group and an azide group, each attached to a different branched linker.
Although an intramolecular reaction (with respect to the same branched linker) is
generally favored over an intermolecular reaction (between two different branched
linkers), the reactivity of the precursors coupled with steric hindrance can make
intermolecular reaction more favorable, especially when there are more candidates
for intermolecular reaction than for intramolecular reaction or when the candidates
for intermolecular reaction is more accessible than those for intramolecular reaction
due to steric hindrance. The efficiency of circularization as used herein refers to
the conversion rate of azide to 1,2,3-triazole on the substrate.
[0050] One way of influencing the efficiency of the click chemistry reaction is to change
the ratio between the alkyne and the azide groups on the substrate. This can be achieved
by reacting the available hydroxyl groups on the second linker with a mixture of an
intermediate for the introduction of the cleavable linker intermediate and a capping
moiety. Such capping moieties can be an acyl, dialkoxyphosphoryl, alkyl, alkoxycarbonyl,
or dialkyaminocarbonyl group, which can react with free hydroxyl groups and make the
hydroxyl group unavailable for the attachment of a cleavable linker and an ensuing
azide-bearing oligonucleotide. The presence of the cap moiety on the branched linker,
not only reduces the steric hindrance associated with oligonucleotides, but it also
provides a higher alkyne to azide ratio to improve the efficiency of circularization
based on the consumption of the azide group. The azide group can have multiple neighboring
alkyne groups to react with when capping moiety is added together with the intermediate
for the introduction of the cleavable linker.
[0051] Finally, the current disclosure allows the introduction of azide-bearing oligonucleotides
and alkynes to the support via individual phosphoramidite monomers, which react with
hydroxyl groups on the surface, instead of introducing both the oligonucleotides and
alkyne on branched linkers.
[0052] Linkers used, including branched linkers and cleavable linkers, can be stable during
oligonucleotide probe synthesis processes such as those discussed herein. Stability
of linkers can allow oligonucleotide probe synthesis without the use of protection-deprotection
steps to preserve the linkers. Linkers can lack reactive species. The absence of reactive
species from the linkers can result in the linkers being inert during a DNA synthesis
process, which can remove the need for protection-deprotection steps. In some cases,
at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, 99.9%, 99.99%, or 99.999% of linkers present prior to DNA synthesis remain
intact after DNA synthesis.
[0053] The probe inversion techniques discussed herein can be conducted in aqueous media.
Avoidance of the use of organic solvents can make such techniques more environmentally
friendly and increase the ease of chemical handling and waste disposal.
[0054] The probe inversion techniques discussed herein can be conducted at a pH of at least
about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,
8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, or 13.5. The probe inversion
techniques discussed herein can be conducted at a pH of at most about 14.0, 13.5,
13.0, 12.5, 12.0, 11.5, 11.0, 10.5, 10.0, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0,
5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, or 0.5. The probe inversion techniques
discussed herein can be conducted at a pH of about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,
4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5,
12.0, 12.5, 13.0, or 13.5. In some cases, the probe inversion techniques discussed
herein can be conducted at or about physiological pH, such as about 7.365 or about
7.5. Conducting reactions at physiological pH can reduce or obviate the need for handling
harsh substances or reaction conditions, and can employ aqueous media.
[0055] The probe inversion techniques discussed herein can be conducted at a temperature
of about 15 °C, 20 °C, 25 °C, 30 °C, or 35 °C. The probe inversion techniques discussed
herein can be conducted at a temperature of at most about 15 °C, 20 °C, 25 °C, 30
°C, or 35 °C. The probe inversion techniques discussed herein can be conducted at
a temperature of at least about 15 °C, 20 °C, 25 °C, 30 °C, or 35 °C. In some cases,
the probe inversion techniques discussed herein can be conducted at or about room
temperature, such as about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24
°C, about 25 °C, about 26 °C, from about 20 °C to about 26 °C, or from about 20 °C
to about 22 °C. Conducting reactions at room temperature can reduce or obviate the
need for handling harsh substances or reaction conditions.
[0056] Releasing truncated probe sequences can increase the percentage of full-length sequences
present in the array. In some cases, at least about 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or 99.999% of probes remaining
bound to the array substrate following a probe inversion process are full-length sequences.
In some cases, a probe inversion process can release at least about 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or 99.999% of
truncated probes bound to the array substrate prior to the probe inversion process.
[0057] The synthesis substrate can comprise different forms or shapes, such as a bead or
a flat array. The synthesis substrate can comprise any suitable material, including
but not limited to glass (e.g., controlled pore glass), silicon, or plastic. Substrates
can comprise polymer coatings or gels, such as a polyacrylamide gel or a PDMS gel.
Gels and coatings can additionally comprise components to modify their physicochemical
properties, for example, hydrophobicity. For example, a polyacrylamide gel or coating
can comprise modified acrylamide monomers in its polymer structure such as ethoxylated
acrylamide monomers, phosphorylcholine acrylamide monomers, betaine acrylamide monomers,
and combinations thereof.
[0058] Inverted probes can provide many advantages over standard non-inverted probes, for
a variety of applications. For example, as discussed above, probe inversion can remove
most or all unwanted truncated sequences, thereby providing a population of inverted
probes containing up to 100% full length sequences. Additionally, inverted probes
can have the 3' OH group free, which can be beneficial for conducting enzymatic reactions
(e.g., single or multiple base extension, ligase reaction). The inverted probes can
also be used for sequencing by synthesis (SBS).
Examples
EXAMPLE 1 - Probe Inversion on Controlled Pore Glass
[0059] Branched linker phosphoramidites having an alkyne moiety were coupled to a dT-derivatized
controlled pore glass (CPG) solid support on a DNA synthesizer using standard DNA
synthesis protocols (see, e.g.,
FIG. 1A). Then, cleavable linkers (CL, see
FIG. 1B) were attached to the branched linkers (Alk, see
FIG. 1B) using standard procedures, followed by synthesis of 19-mer DNA sequences (5'-GGCTGAGTATGTGGTCTAT-3'
(SEQ ID NO: 1)). In the last oligonucleotide coupling step, 5'-bromohexyl phosphoramidite
(Br-Hex, see
FIG. 1B) was incorporated (see
FIG. 1C). DNA synthesis was conducted using an ABI 394 DNA synthesizer using standard DNA
synthesis protocols. Extended coupling time (3 minutes) was used for all linker phosphoramidites
(Alk, CL, and Br-Hex).
[0060] Once DNA synthesis was complete, the 5'-bromo groups were transformed into azide
groups by treating the oligonucleotides on the CPG solid support with NaN
3/NaI in DMF at 65 °C for 1 hour. 13 mg of sodium azide (NaN
3) and 30 mg of sodium iodide (NaI) were dissolved in 2 mL of dry DMF; this solution
was then added to the oligonucleotides loaded on CPG beads in a 2 mL Eppendorf. The
reaction mixture was heated at 65 °C for 1 hour. After cooling down, the reaction
mixture and CPG beads were transferred back to a column. The beads were washed with
water (4 mL) and acetonitrile (6 mL), and then dried under nitrogen.
[0061] Click circularization was then performed using copper catalyzed azide-alkyne cycloaddition
(CuAAC) chemistry. An ascorbic acid stock solution was prepared fresh at a concentration
of 5 mM (e.g., 1.8 mg ascorbic acid in 2.0 mL water). A copper (II)-TBTA stock solution
was prepared at a concentration of 10 mM copper (II)-TBTA in 55% DMSO (e.g., a solution
of 5 mg CuSO
4 in 1 mL water mixed with a solution of 11.6 mg TBTA in 1.1 mL DMSO are mixed). CPG
beads with alkyne-azide modified oligonucleotides were mixed with 55 µL of water in
a pressure-tight vial. 20 µL of 2 M triethylammonium acetate (TEAA) buffer (pH 7.0)
was added to the vial, followed by addition of 95 µL of DMSO. The vial was vortexed.
Then, 20 µL of 5 mM ascorbic acid stock solution was added to the vial, and briefly
vortexed. The solution was degassed by bubbling nitrogen into it for 1 minute. 10
µL of the 10 mM copper (II)-TBTA stock solution was added to the vial. The vial was
then flushed with nitrogen gas, sealed, vortexed thoroughly, and incubated overnight
at room temperature. The final concentrations in the vial were about 0.5 µmol CPG
beads, about 50 vol% DMSO, about 50 vol% water, 0.5 mM ascorbic acid, 0.2 M TEAA buffer,
and 0.5 mM Cu-TBTA complex.
[0062] Only full length oligonucleotides possessing azide functionality were circularized
(see, e.g.,
FIG. 1A). Once click circularization was accomplished, the oligonucleotides were de-protected
and cleaved from the cleavable linker (CL) sites using standard de-protection conditions
(e.g., treatment with NH
4OH at 55 °C for 15 hours).
[0063] The click circularization and probe inversion efficiency were tested by comparing
the HPLC profile of standard non-inverted oligonucleotides (see
FIG. 4A) with the HPLC profile of inverted oligonucleotides (
FIG. 4B). Peaks labeled "1" correspond to non-inverted oligonucleotides, while peaks labeled
"2" correspond to inverted oligonucleotides. HPLC analysis showed that about 70% of
oligonucleotides were inverted under the tested reaction conditions. Increasing the
click circularization reaction time from overnight to 3 days did not increase percentage
of inverted oligonucleotides.
[0064] Reduced efficiency of click circularization may be attributed to steric hindrance
of DNA molecules on dense CPG solid supports. In order to reduce DNA probe density
on the solid support, a mixture of cleavable linkers (CL) and capping phosphoramidites
(CAP, see the insert in
FIG. 4A) was used in different ratios (e.g., 1:0, 1:1, 1:2) immediately after the first phosphoramidite
(branched linker, Alk) coupling. Using a capping phosphoramidite (CAP) directly after
a branched linker (Alk) coupling step can reduce the oligonucleotide density on the
solid support, and can increase the percentage of alkyne which can eventually helped
in click circularization (see
FIGs. 4A-4D, right panels). A 50% reduction in DNA density (CL:CAP 1:1; azide:alkyne ratio 1:2,
FIG. 4C) resulted in inversion of approximately 90% of oligonucleotides. A 66% reduction
in DNA density (CL:CAP 1:2; azide:alkyne ratio 1:3,
FIG. 4D) afforded about 100% inversion of the oligonucleotides. The existence of inverted
oligonucleotides was reaffirmed by comparing the mass spectra of non-inverted (see
FIG. 5A) before click circularization to that of inverted oligonucleotides (see
FIG. 5B) after click circularization.
EXAMPLE 2 - Probe Inversion on Chip
[0065] DNA synthesis on chips (e.g., silanated glass surfaces) was conducted similarly to
the protocols described in
G. H. McGall, F. A. Fidanza, Photolithographic synthesis of arrays. In Methods in
Molecular Biology: DNA Arrays, methods and Protocols; J. B. Rampal, Ed.; Humana Press:
Torowa, NJ, 2001; Vol. 170, 71-101.. DNA density reduction was not deemed necessary for on-chip oligonucleotide inversion,
as inversion efficiency was similar with and without density reduction. In order to
make specific DNA features on the chips, photo-cleavable phosphoramidites were used
instead of standard DMT-phosphoramidites.
[0066] After DNA synthesis, the 5'-bromo groups were converted into azides by treatment
with a mixture of NaN
3 and NaI in DMF at 65 °C for 2 hours. 13 mg of sodium azide (NaN3) and 30 mg of sodium
iodide (NaI) were dissolved in 2 mL of dry DMF; the solution was then added via syringe
to a flow cell containing the chip, and the reaction mixture was heated at 65 °C for
2 hours, with fresh reaction mixture pushed into the flow cell after 1 hour. After
cooling down to room temperature, the reaction mixture was removed and the oligonucleotides
were washed with DMF, water, and acetonitrile, and dried under nitrogen.
[0067] Full length oligonucleotides were then circularized using CuAAC click chemistry (see,
e.g.,
FIG. 1A). The flow cell containing the chip was flushed with nitrogen gas for about 5-10
minutes. A reaction mixture was prepared as follows: 275 µL of water was added to
a 2 mL Eppendorf tube, followed by 100 µL of 0.2 M triethylammonium acetate (TEAA
buffer) at pH 7.0 and 475 µL of DMSO. The mixture was vortexed, 100 µL of 5 mM ascorbic
acid stock solution (as described in Example 1) was added, and the mixture was briefly
vortexed. The solution was then degassed by bubbling with nitrogen gas for 2 minutes.
50 µL of 10 mM copper (II)-TBTA stock solution (as described in Example 1) was added
to the mixture. The reaction mixture was flushed with nitrogen gas for 1 minute, then
added via syringe to the chip under a nitrogen atmosphere. The click circularization
reaction was carried out at room temperature for 24 hours.
[0068] After click circularization, oligonucleotides were de-protected with a 50% EDA/H
2O mixture. EDA treatment was used to de-protect DNA bases and to cleave the 3' end
of the oligonucleotides from the cleavable linker (CL) sites. The chip was incubated
in a 1:1 mixture of ethylene diamine (EDA) and water at room temperature for 7 hours,
with the EDA:H
2O de-protection mixture exchanged with freshly prepared EDA:H
2O mixture every 2 hours. Thereafter chips were washed thoroughly with water and then
stored at 4 °C. This step removed truncated as well as non-circularized oligonucleotides,
allowing only full-length inverted oligonucleotide sequences to remain attached to
the solid surface. The EDA treatment also made the 3'-OH group available for further
enzymatic interventions (see, e.g.,
FIG. 1A).
[0069] To visualize on-chip inverted oligonucleotides, Cy3-labeled DNA sequences, complementary
to the inverted sequences, were used for hybridization (see, e.g.,
FIGs. 6A-6C). Inverted oligonucleotides were hybridized with Cy3 labelled complementary DNA sequences
by placing a 500 nM DNA solution in 4x saline sodium citrate buffer (SSC) on the top
of the chip. The reaction mixture was incubated at 45 °C for 1 hour. Thereafter chip
was washed several times with 4x SSC buffer. The presence of fluorescent signals from
the Cy3 probes after hybridization confirmed the presence of the inverted oligonucleotides
on the chip (see
FIG. 6C). However, no fluorescent signals were observed either from a control chip having
no oligonucleotides (
FIG. 6A) or from a chip where oligonucleotides were de-protected and cleaved without click
circularization (
FIG. 6B). These experiments confirmed that all the on-chip oligonucleotides were inverted.
[0070] The presence of 3'-OH groups on the inverted oligonucleotides was confirmed and visualized
by primer extension using fluorescent dNTPs (see, e.g.,
FIG. 7). The inverted oligonucleotides were first hybridized with DNA templates (5'-CGAACGACGCAATAGACCACATACTCAGCC-3'
(SEQ ID NO: 2)) by incubating the chip with 1 µM of DNA template in 4x SSC at 45 °C
for 1 hour. Thereafter, excess DNA template was washed away from the chip with water.
An extension reaction mixture was prepared with 5 µL of 10x standard Taq buffer (final
amount: 1X), 8 µL of 5 units/µL TaqDNA polymerase solution (final amount: 40 units),
10 µL of 10 µM dNTPs comprising Alexa568-dUTP, dATP, dCTP, and dGTP (final concentration
2 µM), and 27 µL of water. The extension reaction mixture was placed on top of the
chip with hybridized DNA template and incubated at 50 °C for 2 hours in a sealed box
under a humid atmosphere. Following extension, the chip was washed with water prior
to imaging.
[0071] Fluorescent scanning showed strong signals from the extended part of inverted oligonucleotides
(
FIG. 7B), while no fluorescence was noted in the control chip containing no oligonucleotides
(
FIG. 7A). To control for the possibility of nonspecific primer extension at 5'OH terminals,
primer extension was performed on standard non-inverted oligonucleotides having free
5'OH. In this case too, no fluorescent signals were observed before (
FIG. 7C) or after (
FIG. 7D) conducting a primer extension reaction, indicating a lack of primer extension at
the 5'OH terminal.
EXAMPLE 3 - Probe Inversion of ABI Synthesized Oligonucleotide Arrays
[0072] Oligonucleotide arrays were synthesized on an ABI synthesizer and were inverted using
copper-catalyzed azide alkyne cycloaddition reactions as discussed herein. One set
of arrays was fabricated with the three nucleotides at the 3' end having the sequence
-NNT-3' in its different permutations (e.g., -CCT-3', -GCT-3', -ACT-3', -TCT-3', etc.,
as shown in
FIG. 8A). Another set of arrays was fabricated with the two nucleotides at the 3' end having
the sequence -NN-3' in its different permutations (e.g., -CT-3', -GT-3', -AT-3', -TT-3',
etc., as shown in
FIG. 9A).
[0073] Next, the inverted oligonucleotides on the arrays were first hybridized with a DNA
template complementary to those oligonucleotides ending with -TAT-3' or -AT-3'. Inverted
oligonucleotides were hybridized with DNA by incubating the arrays with 1 mL of 1
µM DNA template in 4x SSC at 45 °C for 1 hour. Thereafter excess DNA template was
washed away from the array first with 4X SSC and then with primer extension buffer.
[0074] Primer extension was then carried out with this template using Taq DNA polymerase.
The extension reaction mixture contained Taq polymerase (8 uL, 40 units) and 1 µM
of dNTPs (including Alexa568- dUTP) and was placed on top of the arrays with hybridized
DNA template. The arrays were incubated at 50 °C for 1 hour in a sealed box under
humid atmosphere. Once extension was complete, the arrays were washed with water prior
to imaging.
[0075] Imaging experiments showed signal the regions of the arrays where the 3' end of inverted
oligonucleotides perfectly matched the template DNA (see
FIG. 8B and
FIG. 9B).
EXAMPLE 4 - On-Wafer Probe Inversion
[0076] Oligonucleotide arrays were synthesized on-wafer and were inverted using copper-catalyzed
azide alkyne cycloaddition reactions as discussed herein.
[0077] Next, the inverted oligonucleotide arrays on the wafer were analyzed by hybridizing
alignment marks with a 400 µL of 1 µM Cy3-labeled complementary DNA sequence in 4x
SSC buffer at 55 °C for 1 hour. Thereafter, any excess Cy3-labeled DNA was washed
away from the array with 4x SSC prior to imaging.
FIG. 10 shows an image of alignment marks on a wafer with inverted nucleotides hybridized
with Cy3-labeled complementary DNA.
[0078] Single base primer extension (on the target sequence complementary to the on-chip
inverted DNA sequence) was then carried out by hybridizing the top adapter (FC2')
of the wafer chip with FC2 primer (complementary sequence to FC2') by incubating at
55 °C for 1 hour. The top adapter comprises a region at the top of the inverted sequences.
Excess primer was washed away with 4x SSC, followed by a final wash with incorporation
buffer.
[0079] Illumina incorporation reaction mixture (400 µL), containing DNA polymerase and fluorophore
labelled reversible terminator dNTPs (all four nucleotides were labeled), was placed
on top of the wafer chip which was already hybridized with FC2 primer. The chip was
incubated at 50 °C for 1 hour in a sealed box under humid atmosphere. Once incorporation
was complete, the wafer arrays were washed with incorporation buffer prior to imaging
(see
FIG. 11A-11D). The features which are lighting up in channel 1 show incorporation of dGTP nucleotide
(see
FIG. 11A). Features lighting up in channel 2 show dTTP incorporation (see
FIG. 11B), with dATP incorporation in channel 3 (see
FIG. 11C) and dCTP incorporation in channel 4 (see
FIG. 11D). The barcode region (middle adaptor) of the wafer chip contains an array of sequences
with different base combinations, and an incoming synthesis base could be any of the
four bases (A, T, G, C) at any specific reason. The images shown in
FIG.11A-11D demonstrate the ability to observe which base extension occurred where on the array,
thereby allowing sequencing by synthesis.
[0080] In order to do single base primer extension on the inverted wafer chip sequence,
a 3' blocked complementary sequence, FC2 having an overhang (3'-ACGTTCGAACGTAGCT-5'
(SEQ ID NO: 3)) at 5' position was, hybridized with top adapter of inverted wafer
in the same way as mentioned above. Illumina incorporation reaction mixture (400 mL),
containing DNA polymerase and fluorophore-labelled reversible terminator dNTPs (all
four nucleotides were labeled), was placed on top of the wafer chip which was already
hybridized with primer (3'ddc-FC2-overhang). The chip was then incubated at 50°C for
1 hour in a sealed box under humid atmosphere. Once incorporation was complete, the
wafer arrays were washed with incorporation buffer prior to imaging. The features
were lighted up only in channel 2 which shows incorporation of dTTP (see
FIG. 12B). Since first base in target primer overhang is dATP, the enzyme only incorporated
dTTP at the 3' position of the inverted chip. No signal was seen in any other channels
(see
FIG. 12A, 12C-12D).
[0081] While preferred embodiments of the present invention have been shown and described
herein, it will be obvious to those skilled in the art that such embodiments are provided
by way of example only. Numerous variations, changes, and substitutions will now occur
to those skilled in the art without departing from the invention. It should be understood
that various alternatives to the embodiments of the invention described herein may
be employed in practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures within the scope
of these claims and their equivalents be covered thereby.
SEQUENCE LISTING
[0082]
<110> CENTRILLION TECHNOLOGY HOLDINGS CORPORATION
<120> PROBE INVERSION PROCESS FOR IN SITU SYNTHESIZED PROBE ARRAYS
<130> 38558-711.611
<140>
<141>
<150> 62/293,203
<151> 2016-02-09
<150> 62/206,741
<151> 2015-08-18
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic oligonucleotide"
<400> 1
ggctgagtat gtggtctat 19
<210> 2
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic oligonucleotide"
<400> 2
cgaacgacgc aatagaccac atactcagcc 30
<210> 3
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic oligonucleotide"
<400> 3
tcgatgcaag cttgca 16
<210> 4
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic oligonucleotide"
<220>
<221> modified_base
<222> (17)..(18)
<223> a, c, t, g, unknown or other
<400> 4
ggctgagtat gtggtcnnt 19
<210> 5
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic oligonucleotide"
<400> 5
cgaacgacgc aatagaccac atactcagcc 30
<210> 6
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic oligonucleotide"
<220>
<221> modified_base
<222> (18)..(19)
<223> a, c, t, g, unknown or other
<400> 6
ggctgagtat gtggtctnn 19